Nucleotide sequence of a portion of human chromosome 9 containing a leukocyte interferon gene cluster

.I. Mol. Bid.

(1982)

156. 467-486

Nucleotide Sequence of a Portion of Human Chromosome 9 Containing a Leukocyte Interferon Gene Cluster

IIepartment of Moleculnr Biology Genenteeh, Inc.. 460 Pt. San Bruno Boulevard South Snn Fmncisco. C’al nucleotidr sequence of a 9937 base-pair portion of human chromosome 9, which contains two complete leukocyte interferon genes (LeIF-L and J). the complete intergenic region, and part of a third related possible pseudogene (LeIF-hl). has been determined. The coding regions of the L and .J genes are separated by 4363 nucleotides. The coding regions for the putative L and .J interferons are 96”, homologous and are each surrounded by about 3500 nucleotides of flanking sequences. which are also highly homologous. The L and J genes and their respective flanking sequences comprise a 4000 nucleotide leukocyte interferon gene repeat unit: the L gene repeat unit contains two major insert,ions not present, in the ,J gene repeat unit. The J gene repeat’ unit is flanked by sequence features reminiscent of those found surrounding transposable elements. Both the L and .I gene repeat units are embedded within sequences that are highly repeated in the human genome. Structural features identified within this portion of chromosome 9 may have been important for the generation of this interferon gene cluster.

1. Introduction Int’erferons are a family of proteins that confer viral resistance to many types of mammalian cells by mechanisms that are still poorly understood (Isaacs 8r Lindenmann, 1957 : Raglioni, 1979 ; Stewart, 1979). There are at least’ ten distinct but extremely homologous human leukocyte interferons (LeIF or IFNa: Allen B Fant’es, 1980; Goeddel et 01.. 1981 ; Nagata et nl.. 1981): eight were ident,ified b> DNA sequence analysis of complementary DNA clones derived from human mveloblast messenger RNA (Goeddel et ~1.. 1981) and two were identified b) sequence analysis of genomic DNA clones (Lawn et al.. 198la.b). Fibroblasts appear to contain a single. antigenically distinct interferon (FIF), which is approximatrl> 250/, homologous in amino acid sequence with the leukocyte interferons (Taniguchi et nl., 1980). All of these genes have been localized to human chromosome 9 (Owerbach et al., 1981). Messenger RNA fractionation and in vitro translation experiments indicate t,hat other, perhaps more distantly related fibroblast interferon family members may also exist (Sehgal & Sagar. 1980). 0022-283f@2/110467-20

$03.0170

467

0 1982 Academic Press Inc. (London)

Ltd.

468

A. VLLRICH

E7’ AL

Several of the chromosomal genes for human leukocyte interferons have been identified and isolat’ed (Nagata et al., 1980; Lawn et al.. 198lcc.h), some of which correspond to characterized cDNAsf and others for which a homologous eDNA is yet to be found. In this paper we report the nucleotide sequence of a 9937 base-pair fragment of human chromosome 9 that contains two complete leukocyte interferon genes and a portion of a third related gene. Although no homologous cDNAs have yet been identified for t,hese genes. the coding sequences of two of them are both more closely related to one reported cDNA (LeIF-C) than any of the other seven LeIF vDNAs reported by Goeddel et nC. (1981). These genes and their flanking regions are extremely homologous wit)h each ot’her and contain elements common to other characterized eukaryotic genes. This cluster of LeIF genes bears interestjing structural features which may have been important in the generation of the leukocyte interferon gene family.

2. Materials and Methods (a) I)SA

preparation

Recombinant phage A DNA was prepared by the method of Maniatis et al. (1978) ; plasmid DNA was purified by a modification of the procedure of Clewell (1972). Human spleen (provided by Dr S. Peter) was used as a source of high molecular weight DNA, prepared according to Blin & Stafford (1976). (b) tlestriction

endonuclease

analyni,s

Restriction enzymes, purchased from Bethesda Research Labs or New England Biolabs, were employed in digestion react,ions using conditions specified by the supplier. Reaction products were analyzed on I”i, (w/v) agarose (Seakem) gels or 5 to SyJ polyacrylamide (BioRad) gels. Restriction sites were mapped by analysis of fragments generated by single. double or triple digestions employing multiple restriction endonucleases. In some instances fragment arrangement was determined by Southern hybridization procedures (Southern, 1975) using 32P-labeled (Taylor et al., 1976). cloned cDNA fragments as probes. (v) Sucleotide

sequence determivcation

Overlapping EcoRI and Hind111 fragments of hHLeIF-1 were subcloned into the plasmid pBR322 (Bolivar et al., 1977). further cleaved with RsuI, Sau3A and/or HinfI, and sequenced according to the procedure of Maxam C%(Gilbert (1977). Sequencing reactions were carried out on 5’ end-labeled, separated strands, and yielded overlapping sequences for both DNA strands. Portions of the sequence were further confirmed by the enzymatic method of Sanger et al. (1977) using the single-stranded phage M13mp7 (Messing et al., 1981). (d) Southern

yenomic

analyws

Southern hybridization analyses (Southern, 1975) of genomic DNA were carried out using a vertical gel apparatus according to the procedure described by Owerbach et al. (1981), except that DNA-containing agarose gels were subjected to acid pretreatment (Wahl et al.. 1980). DNA fragments used as hybridization probes in the genomic hybridization experiments were prepared from subclones EcoRI or Hind111 fragments, using various restriction enzymes. The fragments were isolated from preparative polyacrylamide gels by t Abbreviations

used : cl)NA.

complementary

I)NA : kb, 10’ bases or base-pairs as appropriate.

HI’MAN

LEI:KOCYTE

INTERFERON

GENE

CLI’STER

.l.ti!l

electroelution and were 32P-radiolabeled according to the method of Taylor et al. (1976) using [IV 32P]dCTP (New England Nuclear or Amersham). All recombinant DNA experiments were carried out according to guidelines established by the National Institutes of Health.

3. Results Clone hHLeIF-1 was selected from a genomic library prepared from a partial illul and Hoe111 double digest of human fetal liver DNA as described (Lawn et ~1.. 1978). Clone hHLeIF-1 was isolated from a single plaque; DNA obtained from this plaque-purified clone was characterized by restriction endonuclease cleavage in c~onjunction with Southern hybridization (Southern, 1975). Initial Southern hybridization analysis suggested that more than one LeIF gene was present on the cloned human DNA fragment. Determination of the physical map was often complicated by the occurrence of multiple fragments of the same molecular weight after simultaneous digestion of the cloned DNA with two different restriction enzymes. Such ambiguities were clarified by subcloning various restriction fragments of hHLeIF-1 into plasmids. Southern hybridization analysis of various hHLeIF-1 restriction digests using these subcloned fragments as radioactive hybridization probes, in conjunction with data obtained from double digest experiments. allowed us to establish the physical map shown in Figure 1.

Flu. 1. Physical map of the human DNA insert of XHLeIF-1. Restriction endonuclease cleavage sites are designated: EcoRI (I), Hind111 (A), XbaI (0) and BglII (a). Zigzag lines represent the h arms: direction of transcription is indicated by horizontal arrows. Gene regions are boxed, while LeIF coding regions are demarcated with hatched lines.

The cloned human DNA portion of this recombinant X phage is 17% kb in length and contains two complete leukocyte interferon gene regions that are separated from each other by about 4.0 kb (see Fig. 1). Initial hybridization experiments unequivocally detected these two LeIF genes ; however, additional weak bands were occasionally observed (data not shown). As described below, nucleotide sequence determination revealed a third region with considerable homology t’o a LeIF coding sequence. which is located approximately 2.1 kb upstream from one (Lel F-L) of the two LeIF genes (Figs 1 and 2). The third LeIF homologous region was detectable when slightly less stringent hybridization conditions were employed. The nucleotide sequence of the human DNA port’ion of hHLeIF-1 (Fig. 2) was determined by t,he procedure of Maxam & Gilbert (1977), relying upon the physichal map (Fig. 1) and mostly employing subcloned fragments. Rome portions of the intergenic region were confirmed by the enzymatic met,hod of Sanger et al. (1977) using the single-stranded phage M13mp7 (Messing et crl.. 1981).

470

A. ULLRICH

(a) AHLeIF-1

contains

ET AL.

two closely related complete coding regions

Consistent with our hybridization results, two regions within the nucleotide sequence of hHLeIF-1 possess the coding potential for complete interferon proteins. The first of these, located between nucleotides 2262 and 2831 and designated LeIFL, is interrupted by a nucleotide substitution that generates an in phase translation termination codon (TGA). which would halt translation at amino acid position 20 of the presumptive signal peptide of pre-LeIF-L (nucleotide 2335, Fig. 2), three amino acids before the start of the 166 amino acid mature interferon protein sequence. It is not as yet known if this LeIF-L gene is actually transcribed by the cell. Other than this single nucleotide alteration the LeIF-L gene is a perfect member of the LeIF gene family (see Fig. 3). The putat,ive I, gene product. despite containing a premature termination codon would differ by only four amino acids (97.9o/, homologous) when compared wit’h t’he previously described LeIF-C gene product (Goeddel et al.. 1981). The second interferon coding sequence (designated LeIF-J) is located between nucleotides 7 195 and 7764, and is present 4363 nucleotides downstream from LeIFI,, in the same t,ranscriptional orientation. This sequence contains the coding potential for an intact 189 amino acid pre-leukocyte interferon. as shown in Figure 3. The putative J gene product differs by 15 amino acids when compared wit,h LeIF-C (91.9% homology), and by 17 amino acids when compared with LeIFI, (90.90/,, homology).

(b) LeIF-J

and L gene landmarks

When compared with the LeIF-C cDNA sequence (Goeddel et al., 1981), neither the coding regions nor the presumptive 5’ and 3’ untranslated sequences within the mRNA region of the L and J LeIF genes contain intervening sequences, analogous to other LeIF genes (Nagata et al.. 1980; Lawn et al., 1981) and to the FIF gene (Lawn et al., 198lc; Houghton et al., 1981: Tavernier et al.. 1981). With regard to transcriptional signals, mammalian genes often contain a “TATA”-like sequence 23 to 30 nucleotides preceding mRNA cap sites (Goldberg, 1979). Each of the LeIF chromosomal genes examined to date contain the sequence T-A-T-T-T-A-A at this position (Nagata et al., 1980; Lawn et al., 1981a,b). Knowledge of the nucleotide sequence of the very closely related cDNA clone LeIF-C facilitated the designation of transcribed region landmarks: by homology with the cDNA copy of the LeIF-C transcript and location of the T-A-T-T-T-A-A sequence we assigned the mRNA capping sites for LeIF-L to position 2194+3 and for LeIF-J to position 7128f3 (see Fig. 2). As first noted by Benoist et al. (1980), several eukaryotic genes share a sequence of homology about 40 to 70 bases upstream from the “TATA” box. The sequence C-C-A-A-T is found in this region in the p globin gene family (Efstratiadis et al., 1980). Both the L and J interferon genes contain a similar sequence (G-T-AC-A-T-A-A-A and G-T-G-C-A-T-A-A-A. respectively), 65 nucleotides before the T-A-T-T-T-A-A box (Fig. 2). The interferon type of C-C-A-A-T signal is most closely related to that found in the silk fibroin gene of the silk moth (Tsujimoto & Suzuki,

10 CCTTGAGGAGGTACTTC M

20 AGGG

30

40

50

60

70

80

90 100

TCCATGGGAATCCAGAGAATCTACCTGAAAGAGAAGAAATACAGTGACTGTGCTTGGGAGGTTGTCAGAGTGGAATC

ATGAAATCCTTCTCTTCATCAACAGACTTGCAAGGACTGAGAAGTAAGGATGA

GACCTGGGGTCTGCTTTAGTCTTTCTTATTTTCTTCCTCTTCCTAG

CTGTGTGTTTATTTCTTCTTTTCTAGTTCCTTAACTTGTAAAGTTAGTTCATTGGTTTGAGGTCTTTCTTCTTTTTTAATATAAGCTTTTACAGCTTTCA ATTTCCCCTTTAGCTCTGTTTTCACTGCATCACATACGTCTTGGTATGTTGTGTTTTCATTTTCACCTCTCTCAAGATATTTTCTAAGTTCCCCTCTGGT 500

CATCITTTTTTTTTTTGTACTATACTTTAAGTTCCAGGTTAAATGTGCTCAACGTGCAAGTTTGTTACATAGGTATACATTTGCCATGTTGGTTTGCTGC t ACCCATTAACTTGTCATTTACATTAGGTATTTCTCCTCTTACATGTTTATCACATTTAGACATACATTTGTTTTCTTTTTGTTCCCATAAATTAAGATGA AAATCAGACCACTTTTACCTTCTAGGAAAAGTGAAGTGAGAAATATAAATATATTTGCTGTTGTGAATGCCATATAGAACCATTGTATAGTCCATTTAAA AATG

GAATAAATGAGAAATTAGTAAAAACACCACTTACTAAATAGCTGATTTGCTAAAGCAGACCTCATTCCATTTAAGGACTCAGTATCTATAGGGCC L repoot unit L TACTTATACAAAAAAAAACTTCTTACACAAAAAAAAAAAAAAAAAAAAGTTAGAGCCAGAGTTCAGGATCATGCTGAAAGTTATTTCTTTGCATAATAAT 100”

ATTCAATATTTATAAATTTATGAATTTAGAACAAAGATGGTCTTTTTTATTTGATAAGAATTGACTTGGATAGGAACTTCTGAAAACCTTTAGGGAATAT GAACTTCAATGTAAAATGCCAAAAATGATTTAAATCATACTATTTTCTAAGTCATATATGTTTATTGGATTGATACTTCTTTTAAGGGTACAAAAATTAG TTCTCCTAGTGTAAATGAATCTAACATATTACAATAGTTTCTGACTTTCCAACAACTCTATCCAACAAAATTTTATTGCTTAATATACATATTTCTCATT GGGTTTTTTTGTGTATGATATGAGAAGCACTGGTATTGAGTTCATGAAGATAAACAAAATATTTGTAAGACCAATGTTACAAACCTATAGCAAATAGATG ACTGTGATTGGAGGACTTTTTGTCCATTTTTTGCTGGATCTTAAAGTCTTATCACAGTATGTGGCTTTAACCTGCATATCTTTGGGCTGCCATTGACTAT

1500

CTTATAGTTATTAGTTATGTTTGATCCTCAGTTCTTCAGGATGTTTGGTAGACTTTGAGAATTCAATCCAAATAGCTTACATTATATGTTTTATTTCTAC TAAAGTTATTCAATACATCAGTACTTGTGTCAAGTGCTGAAAAGAAAAAAGTTTTGGCAATATCTGGATGAATACTGCAGCTGGTGAATTTACAAATTAT TTTCICATATAAAGCAAAATTCAAACCTTCATACACTAAGAGAAAAATTTTAAAAAATTATTGATTCATATTTTTAGGAGTTTTGAATGATTAGGTAGGT AACTACATTCATATTATTAATGTGTATTATATAGATTTTTATTTTGCATATGTACTTTGATACAAAATTTGCATGAACAAATTATACTAAAAGTTATTCC ACAAATATACTTATCAAATTAAAATAAATGTCAATAGCTTTTAAACTTAGATTTTAGTTTAACTTTTCTGTCATTCTTAACTTTACTTTGAATAAAAAGA

2000

GCAAACTTTGTAGTTTTTATCTGTGAAGTAGAGGTATACGTAATATACATAAATAGATATGCCAAATCTGTGTTACTAAAATTTCATGAAGATTTCAATT AAAAAAAAACCATAAAAGGCTTTGAGTGCAGGTCAAAAAATAGGCAATGATGAAAAAAAACGAAAAACTTTTTAAACACATGGAGAGAGTACATAAAGAAA GCAAAAACAGAGATAGAAA~~~~~~~~~GGGCATTTAGAAAATGGAAATTAGTATGTTCAC~~~~~~~GACCTATGCACAGAGCAAAGTCTTC~GAAAAC CTAGAGGCCGAAGTTCAAGGTTATCCATCTCAAGTAGCCTAGCAATATTTGCAACATCCCA

TGGCCCTGTCCTTTTCTTTACTTATGGCCGTGCTGGTG

CTCAGCTACAAATCCATCTGATCTCTGGGCTGTGATCTGCCTCAGACCCACACCCTGCGTAATAGGAGGGCCTTGATACTCCTGGGACAAATGGGAAGAA TCTClCCTTTCTCCTGCCTGAAGGACAGACATGATTTCCGAATCCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGGCTCAAGCCATCTCTGTCCT

L

2500

CCATGAGATGATCCACCAGACCTTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAAATTTAC CAGCAACTGAATGACLTGGAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAATGAGGACTCCATCCTGGCTGTGAGGAAATACT TCCAAAGAATCACTCTTTATCTAATAGAGAGGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCCCTCTCGTTTTCAACAAA CTTGCAAAAAAGATTAAGGAGGAAGGATTGA

AACTGGTTCAACATGGCAATGATCCTGATTGACTAATACATTATCTCACACTTTCATGAGTTCTTCCA 3000

TTTCAAAGACTCACTTCTATAACCACGACGTGTTGAATCAAAATTTTCAAATGTTTTCAGCAGTGTAAAGAAGTGTCGTGTATACCTGTGCAGGCACTAG TCCTTTACAGATGACCATTCTGATGTCTCTGTTCATCTTTTGTTTAAATATTTATTTAATTATTTTTAAAATTTATGTAATATCATGAGTCGCTTTACAT TGTGCTTAATGTAACAATATATGTTCTTCATATTTAGCCAATATATTAATTTCCTTTTTCATTAAATTTTTACTATACAAAATTTCTTGTGTTTGTTTAT t TCTTTAAGATAAAATGCCAAGGCTGACTTTACAACCTGACTTAAAAATAGATGATTTAATTATGTTACCTATCATAATTTTATTCAAGTTATAAAAATAT ATTTTTTTCTGTACCTGGTTATATGTTCCCTTCAGGATATAAACGTGAACATAAAATATACAGTCCCTGTTCTCTTGTATCTTTGATTTTTTCAGGAAAG AAATCTAAAAACAATAATAATGCTGAATTAATATCAGTGATGCTAACTGCTATAATGTGAGGAAGTAAAAAAACAATGAATTCCTCTTAGCAGAATGTAL

3500

ATTGAGACATATCTGGAAATAAAAGCAGAGATATTCTCTGTAAACTGACTTCAACATGTAATTGAAAATGTACATTGCAAGTCAGATATGTGAATTTGCA GTTTCCAAGGAATACGATATCTGGAAGTTCATAACTGGCAATGGAAAGGACGCAAATGAAGGCTGTCATATGGGGAGCAAGTGGAGAGGGAAAAAAAGAC TTAAACTGGATTCTGACGATCTTCCACCATTAAAGTGTGGGAACAGAAGAGACACAAAGGAAACAGAGGTGGAATACCTTAACATTAGAAGGACAAGAGG ,,,,/,,,,/,,,//,/,,~l////,/l/ GAATGGTGATAAAAGTGTATT GAAAATAAATGTGCTTAGAAAAGGAATCAATAAACTTATGGAAAATGTGAATTAAAACTGAGCACTACAG~AAGAAA~ ,,,,,,,,///////////////////////////////// ATAGATGGCAATGCAGAGCTTACTGAGAGCTGGATTCATAGAATTAATCAGCAGAAGCCATACTGGGGTAGACAGAAGAGTGACTCAGAAAAGAGAAATC ////////////////////////////////////////// AAGATAACACATACAGAAAATGTGAGAAAACTGCCTTTGCAATGGTGGCAAGTAATAAGTTTGGACCCCCCAAAAATGTGGATTATCTTTTATCTGCATA //////////////////////////////////////////

iGAtTiCT.TTiGdAAiGdGGiAiGdATiCtCTtTiTA

AAACGTAGGCAATAGCATTCAGGACATCCGCATGGGCAAAGATTTTATGATGAAATCGCCAAAAGCAACTGCAACCAAAGCTAAAATTGACAAATGGCAT CTAATTAAAGAGATTCTGCACAGCAAAAGAAACTGTCAATCATCACGGTGAACAGGCAACTTACAGAATGGGAGAAAATTTTTACAGCCTACCCAATTGA CAGAGCTCTAATATCCAGAATGGACAAAGAACTTAAACAAATTCACAAAAAAAAAAAAAAGCCCCATCGAAAAGTGGGCAAAGGACATGAACAGACACTT CTCAAAAGTAGACATTTATGTGGCCAATAAACATGAAAAAAGCTCAACATCACTGATCATTAGAGAAATGAAAATCAAAACTGCAATGAGATGCCATCTC (0)

part).

Legend

4000

TAAATGGTGCTGGGAAAACTGGATAGCCATATGCAGAG

AACTGAAACTGGACCCCTTCCTTACACCTTATACAAAAATTAATGCAAGATGGATTAAACGCTTAAATGTAAAACCCAAAACCATAAAAACCCTAGAAGA

PIG:. 2 (first

,I

is on p. 473.

4500

10

20

30

40

50

60

70

80

90

ATGT~ACT~AGAATGGCAATTA~TAAAAAGT~AGGAAA~AA~AGATG~TGGTGAAGCTGTGGAAAAATAGGAAAGCTTTTACACTGTTGGTAGGAATGTA

5000

AATTACTTCAACCATTGTGGAAGACAGTGTGGCGATTCATCAAGGATTTAGAACCAGAAATACCATTAGACCCAGCAATCCTATTCCTGAGTATATACCC AAAGGAATACAAATAATTCTATTATAAAAATACATGCACGTGTATGTTTATTGCAACACTATTTACAATAGCAAAGACAGGAAACCAACCCAAATGCCCA TCAATGATAGAATGGATTAAAAAAATTGTGCT AATTTTTTTAATCCTACTGAAGCTGTAGGATTATTTGGGGGACAGAGACTATTATTCCCTACTTTT repoaf unit L +.3 GGGAATAGTAAATTGTCTGATTCCTACAAACTGTGTGAATTGGAGAGTTTGAATTTCAATATGTGACTCTGTATTTGACACCAGGCTATTTATTTTCTAT TATAACAAAGTAGAGGGAGGATTAGAGATGAAGTCATAAATAGTTAATATAGTGCCAGGCAAAGGATGATATTATGCTGCTCTTGCAACTTGAATCCCCA

5500

GATCTACATGCACCTTAAAAAACTAGAACCCCAGTGGTTTTAGCAGTAAACTAAATGGGCATTACTGATTTTCACTAAAAGCTGAATGGAAATTTTTGTC ATTGTCTATTACAATCCCAAATATGGCCATGATGAAGAAAAACAGCTTCATTCTTGAACACCTTCCATTAG GAAAAAATGAGAAACTAGGAAAAACTC L unit J CACCTACTAAATAGCTGATTTGCTAAAACAGACCTCATTCCATTTAAGGACTCAGTATCTATAGGGCCGAGGCAAACTAACTTTGCCAGAGTTCAGGACC

repeat

ACTCTGAAAGTTAATTCCTTACATAATAATATTCAATATTTATAAATTTATGAATTTAGAACAAAGATGGTCTTTTATATTTGATAAGAATTGACTTGGA TAGGAACTTCTGAAAACCTTTAGGGAATATGAACTTCAATGAAAAATGCCAAAAATGATTTAATTGATAATATTTTCTAAGTCACATATGTTTATTGGAA

6000

TGATACTTCTTCTAAGGGTACAAAAATTAGTTCTCGTAGTGTAAACAAATCTGACATATTGCAAAAGTTTGTAACTCTCCAAGAACTCTATCCAACAAAA TTTCATTGCTTAATATACATCTTTCTCGTTGGGTTTTCTTGTGTATGATATGAGAAGCACTGGTATTGAGTTCATGATGATAAACAAAATATTTGCAAGA TCAACGTTACAAACCTATCCCAAATAGATGACTGTGATTGGAGGACTTTTTGTCAATTTTTTTGCTGGATCTTGAAGTCTTACCACAATATGTGGCTTTA ACCTGCCTACCTTTGTGCTGCCATTGACCATCTTATGGTTATTAGTTATGATTGATCCTCAGTTCTTCAGGATGTTTTGTACACTTTGAGAATTCAATGC AAATAGCCTATATTATATGATTTATTTCTACAAAAGTTATTCAACACATCAGTACTTATGTCAAGTGCTGAAAAGAAAAAAGTGTTGGCAATATCTGGAT

6500

GAATACTGCAGCTAGTGAAGTTTACAAATTATTTTCTCATATAAAGCAAAATTCAAAGCTTCATATACTATGAGAAAATTTTTTTAAAATTGATTCATAT TTCTAGCAGTTTTGAATGATTAGGTATGTAATTACATTCATATTAATGTGTATTATACAGATTTTTATTTTGCATATGTAATTTGAAACAACAAAATTTA CATGAACAAATTACATTAAAAGTTATTCCACAAATATACTTATCTAATTAAACTTAGATTTTAATAGCTTTTAAACTTAGATTTTAGTTTAACTTTTCTG TCATTCTTAACTTACTTTGAATAAAAAGAGCAAACTTCATACTTTTTATCTGTGAAGTAGAGGTATATGTAGAATACCTAAATAGATATGCCAAATCTGT GTTATTAAAATTTCATGAACATTTCAATTAGAAAAAAATACCATAAAAGGCTTTGAGTGCAGGGGAAAAACAGGCAATGATGAAAAAAAAAATGAAAAAC

7000

GTATTTAAACACATGGAGAGAGTGCATAAAGAAAGCAAAAACAGAGATAGAAAGTAAAACTAGGGCATTTAGAAAATGGAAATTAGTATGTTCAC~~~~~ *t*tt**+* v 4bGACCTATGCACAGAGCAAAGTCTCCAGAAAACCTAGAGGCCACGGTTCAAGTTACCCACCTCAGGTAGCCTAGTGATATTTGCAAAATCCC CGGTCCTTTTCTTTACTGATGGTCGTGCTGGTACTCAGCTACAAATCCATCTGCTCTCTGGGCTGTGATCTGCCTCAGACCCACAGCCTGCGTAATAGGA GGGCCTTGATACTCCTGGCACAAATGGGAAGAATCTCTCCTTTCTCCTGCTTGAAGGACAGACATGAATTCAGATTCCCAGAGGAGGAGTTTGATGGCCA

J

CCAGTTCCAGAAGACTCAAGCCATCTCTGTCCTCCATGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGGGAACAG

7500

AGCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATGACCTGGAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGA ATGAGGACTTCATCCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTTTATCTAATGGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGC AGAAATCATGAGATCCTTCTCTTTTTCAACAAACTTGAAAAAAGGATTAAGGAGGAAGGATTG

AACTGGTTCATCATGGAAATGATTCTCATTGACTA

ATGCATCATCTCACACTTTCATGAGTTCTTCCATTTCAAAGACTCACTTCTATAACCACCACAAGTTGAATCAAAATTTCCAAATGTTTTCAGGAGTGTT AAGAAGCATCGTGTTTACCTGTGCAGGCACTAGTCCTTTACAGATGACCATTCTGATGTCTCCTTTCATCTATTTATTTAAATATTTATTTATTTAACTA

8000

TTTTTATTATTTAAATTATTTTTTATGTAATATCATATGTACCTTTACATTGTGGTTAATGTAACAAATATGTTCTTCATATTTAGCCAATATATTAATT TCCTTTTTCATTAAATTTTTACTATACAAAATTTCTTGTGTTTGTTTATTTTTTAAGATTAAATGCCAAGCCTGACTGTATAACCTGACTTAAAAATAGA t TGATTTAAGTAAGTTACCTATCATAATTTTATTCAAGTTATAGAAAAATATATTTTTCTATACCAGGTTATCTGTTGCCTTCATGATATAAACGTGAACA TAAAAAATACAGTTCTTGTTCTCTTGTATCTTTGATTTTTGTCAGGAAAGAAATCTAAAAACAATAATAATGCTGAATTAATATCGGTTATACTAACTGC TGTAATGTGAGGAAGTAAAAAAAAATGAATTCCTCTTAGCAGAACATAGATTAAGAAATGTCTGCAAATAAAAGTAGAGGTACTCTCTATAAACTGACTT

8500

TCAACATGTAATTGAAAATGTACATTGCAAGTCAGATATATGAGTTTGCAGTTTCCAAGGAATATGATATCTGGAAGTTCATAACTAAGCAATGGAAACG CCAAAAATGAAGGCTGTCATGTGGGGACCAAGCAGAGAGGGAAAAAAGACTTAAACTGGATTCTGAGGAACTTCCACTATTAAAGTGGGGGAACAGAAGA CACACAAAGAAAACAGAGGTGGAATACCTTATCATTAGAAGGACGAGAGGGAATGATGATAAAAGTGTATTTGGAGGGAATGATGATAAATGGTGCTGGG AAAACTGGATAGCCATAGGCAGAAAATTGAACCTGGACCACTTCCTTACACCTTATACAAAAATTAACTCAAGATGGATTAAAGACTTAAATGTAAAACC AAAAACCATAAAAACCCTAGAAGAAAACATAGGACACAGGATGGGCAAAGATTTTATGAAGAAATCGCCAAAAGCAACTGCAACAAAAGTTAAATTGACA

9000

AATGCCATTTAATTAGAGAACTTCTGCACAGCAAAAGAAACTACCTATCATCAGAGTGAACAGGCAACCCATAGAATGGGAGAAAATTTTTGCAGTCTAC CCAACTCACACAGGTCTAATATCCAGAATGTACAAAGAACTTAAACAAATTTACAAAAACAAAAAGCCCCATCAAAAAGTGGGTGAAGGATATGAACAGA ATCTTCTCGAAAGTAGACATTTATGCGACCAATAATCATTAAAAAACTCAACATCACTGATCATTAGAGAAATGCAAATCAAAACCACAATGAAATACCA TCTCATGCCACTCACAATGGCAATTATTAAAGAGTCAAAATCAACAGATGCTGGTGAAGCTGTGGAAAAATAGGAAATCTTTTACACTGTTTGTGGGAAT GTAAATTACTTCAACCATTGTGGAAGACAGTGTGGCGATTCATCAAGGTTCTAGAACCAGAAATACCATTTGACCCAGCAATCTCATTACTGAGTATATA CCCAAAGGAATATAAATCGTTCTGTTATAAAATACACGCACGTGTATGTTTATTGCAGCATGTTTACAATAGCAAAGACACAAAACCAACCCAAGTGCCC ATCAATGATAGACTGGATTAAAAAAAATTGTAC TAAAACCTAGGTGACGGATGAGAGTGACCTTGAACTGGCAAGACCAGGTCAGTGCAAGTGACATTG unit J dl GTTTCTTAGATACTCCTCAGCTCCGTGTGGGTCTCCCGATTCTCAGTTCTGCCTGCTGACTTGTTCATTCATTTCCATGGCAGGGCTGCAGTTGGGTCGT

repeat

GCCTGGTTTTTTGGCCTCCCAATGTCTCTTAATTAACTTAATATTACTTGGTAATACTAAAGCAATGATGAATTATGTGCATGGAGTCATTAATCTTCTG TGTAAATATCTCAAACACTGTGTCTTAATTATAGACT

(b)

FTC. 2 (second part). Legend

is

on

p.

453.

9500

HI’MAS

LEI’KOCYTE 5

INTERFEROS 10

GENE 15

17:i

CL17STER 20 25

:

MET MA LEU SEA PHE SER LEU LEU ET MA VAIL LEU VAL LEU SER TYR LYS SER ILE EN0 SER LEU GLY CYS ASP ICT ALA ARG SER PHE SER LEU LEU NET VM VM LEU VM LEU SER TYR LYS SER ILE CYS YR LEU GLY CYS ASP

L J

LEU PRO GLN THR HIS THR LEU ARG ASN ARG AR3 MA LEU ILE LEU LEU GLY GLN KT GLY ARG ILE SER PRO PHE LEU PRO GLN THR HIS SER LEU AW; ASN Affi A16 MA LEU ILE LEU LEU MA GLN bET GLY AP ILE SER PRO PHE

50

SER CYS LEU LYS AS? ARG HIS ASP PHE ARG ILE PRO GLN GLU GLU PHE ASP GLY ASN GLN PHE GLN LYS ALA GLN SER CYS LEU LYS ASP ARG HIS GLU PHE Affi PHE PRO GLU GLU GLU PHE ASP GLY HIS GLN PHE GLN LYS THR GLN

75

: L J

ALA ILE SER VAL LEU HIS GLU FET ILE GLN GLN THR PHE ASN LEU PHE SER THR GLU ASP SER SER ALA MA TRP 100 ALA ILE SER VM LEU HIS GLU EET ILE GLN GLN THR PHE A.9 LEU PHE SER THR GLU ASP SEA SER ALA ALA TRP

L J

GLU GLN SER LEU LEU GLU LYS PHE SER THR GLU ILE TYR GLN GLN LEU ASN ASP LEU GLU ALA CYS VAL ILE GLN 125 GLU GLN SER LEU LEU GLU LYS PHE SER THR GLU LEU TVR GLN GLN LEU ASN ASP LEU GLU ALA CYS VAL ILE GLN

: H

GLU VAL GLY VAL GLU G4U THR PRO LEU liET ASN GLU ASP SER ILE LEU ALA VAL AK LVS TYR PHE GLN ARG ILE 150 GLU VAL GLY VAL GLU GLIJ THR PRO LEU RT ASN GLU Asp PHE ILE LEU ALA VAl Affi LYS TYR PHE GLN AAC ILE LEU ARG ARG TYR PHE XXX GLY SER MT GLY ILE GLU

L 3 M

THR LEU TYR LEU ILE GLU AI% LYS TVA SER PRO CYS AlA TRP GLU VAL VM AK ALA GLU ILE ET ARG SER LEU 175 THR LEU TYR LEU NET GLU LYS LYS TYR YR PRO CYS MA TRP GLU VM VAL ARG MA GLU ILE KT ARG SER PHE ARG ILE TYR LEU LYS 2 LYS LYS TYR SER ASP CYS MA TRP GLU VM VAL Affi VM z SER EN0 AW PRO SER

L J M

SER PHE PR THR A94 LEU GLN LYS ARG LEU ARG AAG LVS ASP SER PHE SER THR ASN LEU LYS LYS GLY LEU Affi Affi LYS ASP LEU HIS GLN GLN THR CYS LYS ASP END GLU VALE PET LYS

VIthank R. M. Lawn for and E. Y\‘. Wong, R. Najarian R. Swanson for support and We especially thank Suzanne to this work.

participation in the initial screening of the human gene bank, and P. H. Seeburg for their contributions. We are grateful to to Jeanne Arch for her expert preparation of the manuscript. Pfeffer for stimulating discussions and valuable contributions

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